Cell-to-cell movement of TMV RNA is temperature-dependent and corresponds to the association of movement protein with microtubules

The movement protein (MP) of tobacco mosaic virus (TMV) is essential for spread of the viral RNA genome from cell to cell. During infection, the MP associates with microtubules, and it has been proposed that the cytoskeleton transports the viral ribonucleoprotein complex from ER sites of synthesis to plasmodesmata through which infection spreads into adjacent cells. However, microtubule association of MP was observed in cells undergoing late infection rather than in cells undergoing early infection at the leading edge of expanding infection sites where virus RNA cell-to-cell spread occurs. Therefore, alternative roles for microtubules in virus infection have been proposed, including a role in MP degradation. To further investigate the role of microtubules in virus pathogenesis, we tested the efficiency of cell-to-cell spread of infection and microtubule association of the MP in response to changes in temperature. We show that the subcellular distribution of MP is temperature-dependent and that a higher efficiency of intercellular transport of virus RNA at elevated temperatures corresponds to an increased association of MP with microtubules early in infection.     

Challenging the role of microtubules in Tobacco mosaic virus movement by drug treatments is disputable

The movement protein (MP) of Tobacco mosaic virus interacts with microtubules during infection. Although this interaction is correlated with the function of MP in the cell-to-cell transport of viral RNA, a direct role of microtubules in the movement process was recently challenged by studies involving the treatment of plants with inhibitors of microtubule polymerization. Here, we report evidence suggesting that such treatments may not efficiently disrupt all microtubules. Thus, results obtained from studies using microtubule inhibitors may have to remain open to interpretation with regard to the involvement of microtubules in viral RNA trafficking.     

Tobacco mosaic virus movement protein interacts with green fluorescent protein-tagged microtubule end-binding protein 1

The targeting of the movement protein (MP) of Tobacco mosaic virus to plasmodesmata involves the actin/endoplasmic reticulum network and does not require an intact microtubule cytoskeleton. Nevertheless, the ability of MP to facilitate the cell-to-cell spread of infection is tightly correlated with interactions of the protein with microtubules, indicating that the microtubule system is involved in the transport of viral RNA. While the MP acts like a microtubule-associated protein able to stabilize microtubules during late infection stages, the protein was also shown to cause the inactivation of the centrosome upon expression in mammalian cells, thus suggesting that MP may interact with factors involved in microtubule attachment, nucleation, or polymerization. To further investigate the interactions of MP with the microtubule system in planta, we expressed the MP in the presence of green fluorescent protein (GFP)-fused microtubule end-binding protein 1a (EB1a) of Arabidopsis (Arabidopsis thaliana; AtEB1a:GFP). The two proteins colocalize and interact in vivo as well as in vitro and exhibit mutual functional interference. These findings suggest that MP interacts with EB1 and that this interaction may play a role in the associations of MP with the microtubule system during infection.     

Tobacco mosaic virus movement protein functions as a structural microtubule-associated protein

The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.     

Validation of microtubule-associated Tobacco mosaic virus RNA movement and involvement of microtubule-aligned particle trafficking

Functional studies of Tobacco mosaic virus (TMV) infection using virus derivatives expressing functional, dysfunctional, and temperature-sensitive movement protein (MP) mutants indicated that the cell-to-cell transport of TMV RNA is functionally correlated with the association of MP with microtubules. However, the role of microtubules in the movement process during early infection remains unclear, since MP accumulates on microtubules rather late in infection and treatment of plants with microtubule-disrupting agents fails to strongly interfere with cell-to-cell movement of TMV RNA. To further test the role of microtubules in TMV cell-to-cell movement, we investigated TMV strain Ni2519, which is temperature-sensitive for movement. We demonstrate that the temperature-sensitive defect in movement is correlated with temperature-sensitive changes in the localization of MP to microtubules. Furthermore, we show that during early phases of recovery from non-permissive conditions, the MP localizes to microtubule-associated particles. Similar particles are found in cells at the leading front of spreading TMV infection sites. Initially mobile, the particles become immobile when MP starts to accumulate along the length of the particle-associated microtubules. Our observations confirm a role for microtubules in the spread of TMV infection and associate this role with microtubule-associated trafficking of MP-containing particles in cells engaged in the cell-to-cell movement of the TMV genome.     

Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of tobacco mosaic virus

Microtubules interact strongly with the viral movement protein (MP) of Tobacco mosaic virus (TMV) and are thought to transport the viral genome between plant cells. We describe a functionally enhanced DNA-shuffled movement protein (MP(R3)) that remained bound to the vertices of the cortical endoplasmic reticulum, showing limited affinity for microtubules. A single amino acid change was shown to confer the MP(R3) phenotype. Disruption of the microtubule cytoskeleton in situ with pharmacological agents, or by silencing of the alpha-tubulin gene, had no significant effect on the spread of TMV vectors expressing wild-type MP (MP(WT)) and did not prevent the accumulation of MP(WT) in plasmodesmata. Thus, cell-to-cell trafficking of TMV can occur independently of microtubules. The MP(R3) phenotype was reproduced when infection sites expressing MP(WT) were treated with a specific proteasome inhibitor, indicating that the degradation of MP(R3) is impaired. We suggest that the improved viral transport functions of MP(R3) arise from evasion of a host degradation pathway.     

Tobacco mosaic virus (TMV) replicase and movement protein function synergistically in facilitating TMV spread by lateral diffusion in the plasmodesmal desmotubule of Nicotiana benthamiana

Virus spread through plasmodesmata (Pd) is mediated by virus-encoded movement proteins (MPs) that modify Pd structure and function. The MP of Tobacco mosaic virus ((TMV)MP) is an endoplasmic reticulum (ER) integral membrane protein that binds viral RNA (vRNA), forming a vRNA:MP:ER complex. It has been hypothesized that (TMV)MP causes Pd to dilate, thus potentiating a cytoskeletal mediated sliding of the vRNA:MP:ER complex through Pd; in the absence of MP, by contrast, the ER cannot move through Pd. An alternate model proposes that cell-to-cell spread takes place by diffusion of the MP:vRNA complex in the ER membranes which traverse Pd. To test these models, we measured the effect of (TMV)MP and replicase expression on cell-to-cell spread of several green fluorescent protein-fused probes: a soluble cytoplasmic protein, two ER lumen proteins, and two ER membrane-bound proteins. Our data support the diffusion model in which a complex that includes ER-embedded MP, vRNA, and other components diffuses in the ER membrane within the Pd driven by the concentration gradient between an infected cell and adjacent noninfected cells. The data also suggest that the virus replicase and MP function together in altering Pd conductivity.     

MPB2C,a microtubule-associated plant protein binds to and interferes with cell-to-cell transport of tobacco mosaic virus movement protein

The movement protein of tobacco mosaic virus, MP30, mediates viral cell-to-cell transport via plasmodesmata. The complex MP30 intra- and intercellular distribution pattern includes localization to the endoplasmic reticulum, cytoplasmic bodies, microtubules, and plasmodesmata and likely requires interaction with plant endogenous factors. We have identified and analyzed an MP30-interacting protein, MPB2C, from the host plant Nicotiana tabacum. MPB2C constitutes a previously uncharacterized microtubule-associated protein that binds to and colocalizes with MP30 at microtubules. In vivo studies indicate that MPB2C mediates accumulation of MP30 at microtubules and interferes with MP30 cell-to-cell movement. In contrast, intercellular transport of a functionally enhanced MP30 mutant, which does not accumulate and colocalize with MP30 at microtubules, is not impaired by MPB2C. Together, these data support the concept that MPB2C is not required for MP30 cell-to-cell movement but may act as a negative effector of MP30 cell-to-cell transport activity.     

Cellular targets of functional and dysfunctional mutants of tobacco mosaic virus movement protein fused to green fluorescent protein

Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.     

Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein.

The mechanisms that direct positive-stranded RNA virus replication complexes to plant and animal cellular membranes are poorly understood. We describe a specific interaction between a replication protein of an RNA plant virus and membranes in vitro and in live cells. The tobacco etch virus (TEV) 6 kDa protein associated with membranes as an integral protein via a central 19 amino acid hydrophobic domain. In the presence or absence of other viral proteins, fluorescent fusion proteins containing the 6 kDa protein associated with large vesicular compartments derived from the endoplasmic reticulum (ER). Infection by TEV was associated with a collapse of the ER network into a series of discrete aggregated structures. Viral RNA replication complexes from infected cells were also associated with ER-like membranes. Targeting of TEV RNA replication complexes to membranous sites of replication is proposed to involve post-translational interactions between the 6 kDa protein and the ER.     

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.     

Role of P30 in replication and spread of TMV

The P30 movement protein (MP) of tobacco mosaic virus is essential for distribution of sites of replication within infected cells and for cell–cell spread of infection. MP is an integral membrane protein and in early and mid-stages of infection causes severe disruption of the cortical endoplasmic reticulum (ER). MP also associates with microtubules, and in late stages is targeted for degradation by the 26S proteosome. During these stages, the ER regains its normal pre-infection configuration. Viral RNA is associated with ER and microtubules in the presence of MP. The MP is phosphorylated and mutation of the phosphorylated amino acid reduced association of MP with the ER, plasmodesmata, and microtubules, and altered the stability of the MP. The nature of the association of MP with vRNA and ER and microtubules, and the role of phosphorylation of MP in each of these functions, if any, remains to be determined.     

Intramolecular complementing mutations in tobacco mosaic virus movement protein confirm a role for microtubule association in viral RNA transport

The movement protein (MP) of Tobacco mosaic virus (TMV) facilitates the cell-to-cell transport of the viral RNA genome through plasmodesmata (Pd). A previous report described the functional reversion of a dysfunctional mutation in MP (Pro81Ser) by two additional amino acid substitution mutations (Thr104Ile and Arg167Lys). To further explore the mechanism underlying this intramolecular complementation event, the mutations were introduced into a virus derivative expressing the MP as a fusion to green fluorescent protein (GFP). Microscopic analysis of infected protoplasts and of infection sites in leaves of MP-transgenic Nicotiana benthamiana indicates that MP(P81S)-GFP and MP(P81S;T104I;R167K)-GFP differ in subcellular distribution. MP(P81S)-GFP lacks specific sites of accumulation in protoplasts and, in epidermal cells, exclusively localizes to Pd. MP(P81S;T104I;R167K)-GFP, in contrast, in addition localizes to inclusion bodies and microtubules and thus exhibits a subcellular localization pattern that is similar, if not identical, to the pattern reported for wild-type MP-GFP. Since accumulation of MP to inclusion bodies is not required for function, these observations confirm a role for microtubules in TMV RNA cell-to-cell transport.     

NTH201, a novel class II KNOTTED1-like protein, facilitates the cell-to-cell movement of Tobacco mosaic virus in tobacco

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.     

Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA

Little is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.     

A dysfunctional movement protein of tobacco mosaic virus interferes with targeting of wild-type movement protein to microtubules.

The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent.     

A novel direct interaction of endoplasmic reticulum with microtubules.

The positioning and dynamics of organelles in eukaryotic cells critically depend on membrane-cytoskeleton interactions. Motor proteins play an important role in the directed movement of organelle membranes along microtubules, but the basic mechanism by which membranes stably interact with the microtubule cytoskeleton is largely unknown. Here we report that p63, an integral membrane protein of the reticular subdomain of the rough endoplasmic reticulum (ER), binds microtubules in vivo and in vitro. Overexpression of p63 in cell culture led to a striking rearrangement of the ER and to concomitant bundling of microtubules along the altered ER. Mutational analysis of the cytoplasmic domain of p63 revealed two determinants responsible for these changes: an ER rearrangement determinant near the N-terminus and a central microtubule-binding region. The two determinants function independently of one another as indicated by deletion experiments. A peptide corresponding to the cytoplasmic tail of p63 promoted microtubule polymerization in vitro. p63 is the first identified integral membrane protein that can link a membrane organelle directly to microtubules. By doing so, it may contribute to the positioning of the ER along microtubules.     

Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles

All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes.     

Interaction of Sesbania mosaic virus movement protein with VPg and P10: implication to specificity of genome recognition

Sesbania mosaic virus (SeMV) is a single strand positive-sense RNA plant virus that belongs to the genus Sobemovirus. The mechanism of cell-to-cell movement in sobemoviruses has not been well studied. With a view to identify the viral encoded ancillary proteins of SeMV that may assist in cell-to-cell movement of the virus, all the proteins encoded by SeMV genome were cloned into yeast Matchmaker system 3 and interaction studies were performed. Two proteins namely, viral protein genome linked (VPg) and a 10-kDa protein (P10) c v gft encoded by OFR 2a, were identified as possible interacting partners in addition to the viral coat protein (CP). Further characterization of these interactions revealed that the movement protein (MP) recognizes cognate RNA through interaction with VPg, which is covalently linked to the 5' end of the RNA. Analysis of the deletion mutants delineated the domains of MP involved in the interaction with VPg and P10. This study implicates for the first time that VPg might play an important role in specific recognition of viral genome by MP in SeMV and shed light on the possible role of P10 in the viral movement.